Endohedrally Functionalized Heteroleptic Coordination Cages for Phosphate Ester Binding

Abstract Metallosupramolecular hosts of nanoscopic dimensions, which are able to serve as selective receptors and catalysts, are usually composed of only one type of organic ligand, restricting diversity in terms of cavity shape and functional group decoration. We report a series of heteroleptic [Pd2A2B2] coordination cages that self‐assemble from a library of shape complementary bis‐monodentate ligands in a non‐statistical fashion. Ligands A feature an inward pointing NH function, able to engage in hydrogen bonding and amenable to being functionalized with amide and alkyl substituents. Ligands B comprise tricyclic aromatic backbones of different shape and electronic situation. The obtained heteroleptic coordination cages were investigated for their ability to bind phosphate diesters as guests. All‐atom molecular dynamics (MD) simulations in explicit solvent were conducted to understand the mechanistic relationships behind the experimentally determined guest affinities.


NMR Spectroscopy
NMR spectroscopic data was measured on the spectrometers Bruker AV 500 Avance NEO, Bruker AV 400 Avance III HD NanoBay, AV 500 Avance III HD, AV 600 Avance III HD, AV 700 Avance III HD and Agilent Technologies DD2 500 MHz. For 1 H and 13 C NMR spectra, chemical shifts were calibrated to the solvent lock signal. For 31 P NMR spectra, 85% H3PO4 in H2O ( 31 P, 0 ppm) was used as external standard. Chemical shifts δ are given in ppm, coupling constants J in Hz. All spectra were recorded in standard 5 mm NMR tubes at 25 °C, if not mentioned otherwise. 13 C{ 1 H} NMR spectra were processed using automatic BLP (backwards linear prediction, "cryoproc1d") to optimize the baseline. Due to overlapping signals and low signal-to-noise ratio in the aromatic regions not every 13 C signal of the coordination cages could be observed. 1 H DOSY NMR spectra were recorded with a dstebpgp3s pulse sequence with diffusion delays D20 of 0.08 s and gradient powers P30 of 2500 to 3000 µs. [1,2] T1 analyses of the corresponding signals in the 1D spectra were performed to obtain the diffusion coefficients D using the STEJSKAL-TANNER-Equation. [3,4] Hydrodynamic radii rH were calculated using the STOKES-EINSTEIN-Equation. [5] 1.2 X-Ray single crystal structure determination Synchrotron beamline P11@DESY: Single crystal X-ray diffraction data was collected at macromolecular beamline P11, Petra III, DESY (a member of the Helmholtz Association, HGF), Hamburg, Germany. Samples were mounted using the Stäubli TX60L robotic arm. A wavelength of l = 0.6889 Å was chosen using a liquid N2 cooled double crystal monochromator. Single crystal X-ray diffraction data was collected at 100(2) K on a single axis goniometer, equipped with an Oxford Cryostream 800 device and an Eiger 2 12M detector.

Mass spectrometry and ion mobility measurements
Mass spectrometry and trapped ion mobility data were measured on Bruker ESI-timsTOF (electrospray ionization-trapped ion mobility-time of flight) and Bruker compact high-resolution LC mass spectrometers (positive/negative mode). For calibration of the TIMS and TOF devices, Agilent ESI-Low Concentration Tuning Mix was used. All measured and calculated values are given in m/z.

GPC
Recycling gel permeation chromatography was performed on Japan Analytical Industry NEXT and LaboACE instruments using JAIGEL 1-HH and 2-HH columns, 20 mm x 600 mm, flowrate 7 mL/min.

UV/Vis spectroscopy
UV vis spectra were recorded on a DAD HP-8453 UV-Vis spectrometer.

CD spectroscopy
Circular dichroism spectra were recorded on an Applied Photophysis qCD Chirascan CD spectrometer with a temperature-controlled cuvette holder.

Experimental Procedures
Where necessary, experiments were performed under argon atmosphere using standard Schlenk techniques. Chemicals and standard solvents were purchased from Sigma Aldrich, Acros Organics, Carl Roth, TCI Europe, VWR, ABCR or other suppliers and used as received, if not mentioned differently. Dry solvents were purchased or purified and dried over absorbent-filled columns on a GS-Systems solvent purification system (SPS
2,7-dibromo-9H-carbazole (1.00 g, 3.08 mmol, 1.0 eq) and ethyl-N-methylcarbamate (1.27 g, 12.31 mmol, 4.0 eq) were dissolved in freshly distilled phosphoryl trichloride (20 mL) and heated to 85 °C for 24 h. The reaction mixture was cooled to rt and diluted with ice-cold water. Saturated NaHCO3 solution was added until the mixture was pH-neutral. The reaction mixture was extracted with DCM and the combined organic layers were washed with water and brine and dried over MgSO4. The solvent was removed in vacuo and the crude product was purified via column chromatography (pentane/ethyl acetate 9:1) to obtain 3 as a white solid (0.29 g, 0.75 mmol, 24%).

Guest Titration Experiments
1 H NMR Titration experiments were carried out in the following way: The guest solution (15 mM in DMSO-d6) was added to the host solution (0.7 mM in DMSO-d6) in steps of 0.2 eq (until reaching 2 eq in total, then 2.5, 3.0, 3.5, 4.0 and 5.0 eq, if not mentioned otherwise). After adding, the sample was shaken briefly and measured directly. Owing to clarity, only every second spectrum is shown in the following figures.

Titration of G 3 to Pd2L 2 2L A 2 in DMF-d7
Figure S124: Stacked partial 1 H NMR spectra (500 MHz, 298 K, DMF-d7) of G 3 @Pd2L 2 2L A 2. Since it was not possible to determine binding constants for the aliphatic guest molecules (see discussion in main text and above), we performed guest competition experiments to test for the relative binding affinity of aliphatic G 4 compared to aromatic G 3 , qualitatively. Therefore, first 1 eq of G 4 was added to a solution of Pd2L 1 2L A 2, followed by 1 eq of G 3 . NMR spectra were recorded immediately and 24 and 48 h after addition of G 3 . As a result, compared to the spectrum of pure G 3 @Pd2L 1 2L A 2, the aliphatic guest was found to be fully replaced by the aromatic one, indicating that G 3 has a higher affinity to the host than G 4 (compare a similar trend in the MD simulation results for G 3 @Pd2L 2 2L A 2 vs. G 4 @Pd2L 2
Next, the order of guest addition was exchanged. First, 1 eq of G 3 was added, yielding the host-guest complex G 3 @Pd2L 1 2L A 2, followed by addition of 1 eq of G 4 , leading to only a slight further downfield shift of the inward pointing NH proton, expected for a situation where G 3 is the stronger binding guest (the additional downfield shift is explainable by the doubling of the phosphate guest concentration in this experiment, regardless of substituent). Again, this experiment confirms that G 3 is the stronger binding guest. Figure S126: Stacked partial 1 H NMR spectra (500 MHz, 298 K, DMSO-d6) of (from bottom to top) Pd2L 1 2L A 2, 1 eq G 3 to Pd2L 1 2L A 2, 1 eq G 4 to G 3 @Pd2L 1 2L A 2, the same after 24 h, after 48 h and pure G 3 @Pd2L 1 2L A 2 for comparison (for spectrum of pure G 4 @Pd2L 1 2L A 2 see 2 nd spectrum from bottom in Fig. S126).

5
Single-crystal X-ray structure analysis were studied using single-crystal X-ray crystallography. Due to very thin plate-shaped crystals, the analysis was hampered by the limited scattering power of the samples not allowing to reach the desired (sub-)atomic resolution using our modern microfocussed X-ray in-house CuKα source. Gaining detailed structural insight required cryogenic crystal handling and highly brilliant synchrotron radiation. Hence, diffraction data of all three supramolecular assemblies was collected during two beamtime shifts at macromolecular synchrotron beamline P11, PETRA III, DESY. [8] Counterion and solvent flexibility required carefully adapted macromolecular refinement protocols employing geometrical restraint dictionaries, similarity restraints and restraints for anisotropic displacement parameters (ADPs).

Data collection and refinement details of [Pd2L 1 2L A 2], ap136d
Single crystals were grown by slow diffusion of toluene into a solution of [Pd2L 1 2L A 2] in dimethylformamide (DMF). A single crystal of [Pd2L 1 2L A 2] in mother liquor was pipetted onto a glass slide containing NVH oil. To avoid cracking of the crystal, the crystal was quickly mounted onto a 0.06 mm nylon loop and immediately flash cooled in liquid nitrogen. Crystals were stored at cryogenic temperature in dry shippers, in which they were safely transported to macromolecular beamline P11 at Petra III, [8] DESY, Germany. A wavelength of λ = 0.6888 Å was chosen using a liquid N2 cooled double crystal monochromator. Single crystal X-ray diffraction data was collected at 100(2) K on a single axis goniometer, equipped with an Oxford Cryostream 800 open flow cooling device and an Eiger 2 12M detector. 3600 diffraction images were collected in a 360° φ sweep at a detector distance of 154 mm, 100% filter transmission, 0.1° step width and 50 ms exposure time per image. Data integration and reduction were undertaken using XDS. [9] The structure was solved by intrinsic phasing/direct methods using SHELXT [10] and refined with SHELXL [11] using 22 cpu cores for full-matrix least-squares routines on F 2 and ShelXle [12] as a graphical user interface and the DSR program plugin was employed for modeling. [13,14] The asymmetric unit contains a full cage. Two of the four co-crystallised tetrafluoroborate was modelled with two discrete positions refining their occupancy factor to 78:22 using a free variable. Four of the five co-crystallised toluene molecules as well as one of the two co-crystallised DMF molecules were modelled disordered refining their occupancy factor with a free variable for to 66%:34% for Toluene in residue 21-22 and 78%:22% for DMF in residue 11-12. An additional free variable was refined to occupancy of 67%:53% for toluene in residues 19-20, 24-25, 28-29 as well as tetrafluoroborate in residues 9-10, all of which are in closed proximity to each other. Despite reaching 0.74 Å resolution, disorder and poor crystal quality required stereochemical restraints to be employed for ensuring a sensible geometry of the organic part of the structure. Stereochemical restraints for the ligands L 1 (residue class ICZ) and L A (residue class MPR) and cocrystallised toluene (residue class TOL) and dimethylformamide (residue class DMF) solvent molecules were generated by the GRADE program using the GRADE Web Server (http://grade.globalphasing.org) and applied in the refinement. A GRADE dictionary for SHELXL contains target values and standard deviations for 1,2-distances (DFIX) and 1,3-distances (DANG), as well as restraints for planar groups (FLAT). All displacements for non-hydrogen atoms were refined anisotropically. The refinement of ADP's for carbon, nitrogen, oxygen, boron and fluorine atoms was enabled by a combination of similarity restraints (SIMU) with lowered standard deviation of [0.02 0.04] and rigid bond restraints (RIGU). [15] For disordered toluene molecules in residues 24-25 additional ISOR restraints were applied.

Data collection and refinement details of [G 3 @Pd2L 1 2L A 2], ap214
Single crystals were grown by slow diffusion of diethylether into a solution of [G 3 @Pd2L 1 2L A 2] in dimethylformamide (DMF). A single crystal of [G 3 @Pd2L 1 2L A 2] in mother liquor was pipetted onto a glass slide containing NVH oil. To avoid cracking of the crystal, the crystal was quickly mounted onto a 0.3 mm nylon loop and immediately flash cooled in liquid nitrogen. Crystals were stored at cryogenic temperature in dry shippers, in which they were safely transported to macromolecular beamline P11 at Petra III [8] , DESY, Germany. A wavelength of λ = 0.6888 Å was chosen using a liquid N2 cooled double crystal monochromator. Single-crystal X-ray diffraction data was collected at 100(2) K on a single axis goniometer, equipped with an Oxford Cryostream 800 open flow cooling device and an Eiger 2 12M detector. 3600 diffraction images were collected in a 360° φ sweep at a detector distance of 154 mm, 100% filter transmission, 0.1° step width and 10 ms exposure time per image. Data integration and reduction were undertaken using XDS. [9] The structure was solved by intrinsic phasing/direct methods using SHELXT [10] and refined with SHELXL [11] using 22 cpu cores for full-matrix least-squares routines on F 2 and ShelXle [12] as a graphical user interface and the DSR program plugin was employed for modeling. [13,14] The asymmetric unit contains half a cage, one diphenylphosphate (residue class POB), one tetrafluoroborate counter ion (residue class BF4), three diethylether as well as one dimethylformamide solvent molecules. One of the three co-crystallised diethylether solvent molecules is close to a special position (2-fold axis) and was therefore modelled with negative PART and a fixed occupancy factor of 50%. Despite reaching 0.74 Å resolution, disorder and poor crystal quality required stereochemical restraints to be employed for ensuring a sensible geometry of the solvent part of the structure. Stereochemical restraints for the co-crystallised diethyether (residue class ETO) and dimethylformamide (residue class DMF) solvent molecules were generated by the GRADE program using the GRADE Web Server (http://grade.globalphasing.org) and applied in the refinement. A GRADE dictionary for SHELXL contains target values and standard deviations for 1,2-distances (DFIX) and 1,3-distances (DANG), as well as restraints for planar groups (FLAT). All displacements for non-hydrogen atoms were refined anisotropically. The refinement of ADP's for carbon, nitrogen, oxygen, boron and fluorine atoms of dimethylformamide and diethylether in residues classes DMF and ETO was enabled by a combination of similarity restraints (SIMU) and rigid bond restraints (RIGU). [15] Rigid bond restraints (RIGU) were also employed for the phosphate guest (G 3 ) in residue class POB.

Data collection and refinement details of [G 5 @Pd2L 1 2L A 2], ap217_sq
Single crystals were grown by slow diffusion of diethylether into a solution of [G 5 @Pd2L 1 2L A 2] in dimethylformamide (DMF). A single crystal of [G 5 @Pd2L 1 2L A 2] in mother liquor was pipetted onto a glass slide containing NVH oil. To avoid cracking of the crystal, the crystal was quickly mounted onto a 0.1 mm nylon loop and immediately flash cooled in liquid nitrogen. Crystals were stored at cryogenic temperature in dry shippers, in which they were safely transported to macromolecular beamline P11 at Petra III, [8] DESY, Germany. A wavelength of λ = 0.77491 Å was chosen using a liquid N2 cooled double crystal monochromator. Single crystal X-ray diffraction data was collected at 100(2) K on a single axis goniometer, equipped with an Oxford Cryostream 800 open flow cooling device and an Eiger 2 12M detector. 3600 diffraction images were collected in a 360° φ sweep at a detector distance of 154 mm, 100% filter transmission, 0.1° step width and 10 ms exposure time per image. Data integration and reduction were undertaken using XDS. [9] The structure was solved by intrinsic phasing/direct methods using SHELXT [10] and refined with SHELXL [11] using 22 cpu cores for full-matrix least-squares routines on F 2 and ShelXle [12] as a graphical user interface and the DSR program plugin was employed for modeling. [13,14] The asymmetric unit contains half a cage, one ditoloylphosphate (residue class POT), one tetrafluoroborate counter ion (residue class BF4) and a co-crystallised dimethylformamide solvent molecule. Despite reaching 0.83 Å resolution, poor crystal quality required stereochemical restraints to be employed for ensuring a sensible geometry of the solvent part of the structure. Stereochemical restraints for the co-crystallised dimethylformamide (residue class DMF) solvent molecule were generated by the GRADE program using the GRADE Web Server (http://grade.globalphasing.org) and applied in the refinement. A GRADE dictionary for SHELXL contains target values and standard deviations for 1,2-distances (DFIX) and 1,3-distances (DANG), as well as restraints for planar groups (FLAT). All displacements for non-hydrogen atoms were refined anisotropically. The refinement of ADP's for carbon, nitrogen, oxygen, boron and fluorine atoms was enabled by a combination of similarity restraints (SIMU) and rigid bond restraints (RIGU). [15] . The contribution of the electron density from disordered solvent molecules, which could not be modeled with discrete atomic positions were handled using the SQUEEZE [16] routine in PLATON. [17] The solvent mask file (.fab) computed by PLATON were included in the SHELXL refinement via the ABIN instruction leaving the measured intensities untouched.

Ion Mobility Measurements
Ion mobility measurements were performed on a Bruker timsTOF instrument combining a trapped ion mobility (TIMS) with a time-of-flight (TOF) mass spectrometer in one instrument.
In contrast to the conventional drift tube method to determine mobility data, where ions are carried by an electric field through a stationary drift gas, the TIMS method is based on an electric field ramp to hold ions in place against a carrier gas pushing them in the direction of the analyzer. Consequently, larger sized ions that experience more carrier gas impacts leave the TIMS units first and smaller ions elute later. This method offers a much higher mobility resolution despite a smaller device size.
Measurement: After the generation of ions by electrospray ionisation (ESI, analyte concentration: 0.07 mM, solvent: acetonitrile, capillary voltage: 3600V, end plate offset voltage: 500V, nebulizer gas pressure: 0.3 bar, dry gas flow rate: 3.0 L/min, dry temperature: 75 °C) the desired ions were orthogonally deflected into the TIMS cell consisting of an entrance funnel, the TIMS analyser (carrier gas: N2, temperature: 305 K, entrance pressure: 2.55 mbar, exit pressure: 0.89 mbar, IMS imeX ramp end: 1.92 1/K0, IMS imeX ramp start: 0.54 1/K0) and an exit funnel. As a result, the ions are stationary trapped. After accumulation (accumulation time: 10 ms), a stepwise reduction of the electric field strength leads to a release of ion packages separated by their mobility. After a subsequent focussing, the separated ions are transferred to the TOF-analyser.
The ion mobility K was directly calculated from the trapping electric field strength E and the velocity of the carrier gas stream vg via where ze is the ion charge, kB is the BOLTZMANN constant, µ is the reduced mass of analyte and carrier gas and N0 is the number density of the neutral gas. [18][19][20] For calibration of both the TIMS and TOF analysers, commercially available Agilent ESI tuning mix was used. The instrument was calibrated before each measurement, including each change in the ion mobility resolution mode ("imeX" settings: survey, detect or ultra).    Taking a close look at the measured ion mobility spectra, we observe that the signals are mostly gaussian-shaped or have slight shoulders. For some of the species with bound guest we observe two prominent mobility signals, indicating different co-conformers of the host-guest complexes in the gas phase, probably due to different positions of the guest inside the anisotropic cage. However, we were not able to differentiate between different conformers by means of theoretical CCS calculations (compare below). In all cases, the measured mobilities of the host-guest complexes are close to the mobilities of the hosts containing only a BF4anion, indicating encapsulation inside the cavity (withdepending on the guest -slight protrusion of guest features, i.e. the phosphate's alkyl or aryl substituents outside the cage's apertures).

Modeling and Theoretical Collisional Cross Sections calculations (CCS)
The host-guest systems were optimized using the quantum chemistry software package ORCA (version 5.0.2), [21] by DFT methods on a PBE/def2-SVP level of theory. The theoretical collisional cross sections ( Theo CCSN2) of the optimized models were then calculated with Collidoscope (version 1.4). [22] The number of energy states was set to 16 and the temperature to 303 K. The used CM5 point charges [23] were calculated using the xtb software (version 6.4.1) and the semiempirical model GFN1-xTB. [24]  For this system we observe a relatively large average deviation, around +7.5%, of the theoretical value compared to the experimental data. This could be due to neglecting dynamic cage motions in the CCS calculation technique and also due to the unique, funnel-like shape of the cage that may be problematic in the used trajectory method. The error is especially large for the [Pd2L 1 2L C 2 + X] 3+ species, which may be attributed to the two hexyl side chains, considered straight in the DFT models but probably adopting more folded conformations in the gas phase. The slight increase of the CCS value by replacing a BF4anion with a guest, as observed experimentally, could be reproduced in the computations. As discussed in the main text, considering a guest binding mode outside of the cage would lead to very high deviations between measured and calculated CCS values (see 6 th entry in Table S2), which is why we assume the guest to be always inside the cage during the TIMS measurement. UV/Vis and CD spectroscopy Figure S137: UV/vis spectra of Pd2L 1 2L A 2 (green, c = 0.07 mM), G 6 @Pd2L 1 2L A 2 (1 eq red, 2 eq blue, c = 0.07 mM) and G 6 (black, c = 0.07 mM). Figure S138: CD spectra of Pd2L 1 2L A 2 (green, c = 0.07 mM), G 6 @Pd2L 1 2L A 2 (1 eq red, 2 eq blue, c = 0.07 mM) and G 6 (black, c = 0.07 mM).
A small CD signal in the absorption range of the host-guest complex > 400 nm was observed upon addition of the chiral guest to the achiral host, indicating a certain degree of chirality transfer from the guest to the host. Figure S139: Overlay of UV/Vis (solid lines) and CD (dashed line) spectra of Pd2L 1 2L A 2 (green, c = 0.07 mM), G 6 @Pd2L 1 2L A 2 (1 eq red, c = 0.07 mM) and G 6 (black, c = 0.7 mM), showing that the emerging CD band > 400 nm belongs exclusively to a host absorption.

Molecular Dynamics Simulations
Initial general AMBER force field (GAFF) parameters [25] and topologies for the heteroleptic cages [Pd2L 1 4+ and [Pd2L 2 4+ as well as for the diphenylphosphate guest G 3 were generated with the CHIMERA software. [26] Parameters for the bonds and angles involving Pd were taken from our previous work, [27] and Lennard-Jones (6,12) parameters for the palladium were taken from Yoneya et al. [28] Atomic partial charges for host and guest were calculated with the ESP method using the DFT functional B3LYP [29] with the 6-31G* all-electron basis set for all atoms except Pd, for which the Stuttgart-Dresden (SDD) pseudopotentials [30] were used. After force field parametrisation, the cage and one guest molecule were placed randomly in a periodic simulation box with a volume of ca. 67 nm 3 and solvated in DMSO, for which parameters were taken from the work of van der Spoel et al. [31] Three tetrafluoroborate anions were added in the solvent to keep the overall charge of the simulation box zero. The system was energy minimized using steepest descent and then equilibrated at 298 K in a 500 ps NVT simulation, followed by 500 ps NPT simulation. The final production runs were carried out in the NPT ensemble. The LINCS algorithm [32] was used to constrain bond lengths involving H-atoms, allowing to integrate the equations of motion with 2 fs time steps using the leapfrog integrator. Temperature was kept constant at 298 K with the velocity-rescale thermostat of Bussi and coworkers [33] with a coupling time constant of 0.1 ps. To maintain constant 1 bar pressure, the Berendsen barostat was used with a coupling time constant of 2 ps. Shortrange Lennard-Jones (6,12) and Coulomb interactions were treated with a buffered Verlet pair list with a cut-off of 1.0 nm. Long-range Coulomb interactions were treated with the PME algorithm with 0.12 nm grid spacing. Analytical corrections to energy and pressure were applied to compensate for the truncation of the Lennard-Jones interactions. In the production runs, unbiased MD simulations of the host-guest system were performed. For each of the two cages investigated, ten independent simulations, each 5 μs long, were carried out, yielding a total sampling time of 100 μs. These extended simulation times enabled the observation of multiple spontaneous binding and unbinding events. The total number of binding/unbinding events observed during the simulations was 32 and 70 for the cages [Pd2L 1 and [Pd2L 2 4+ , respectively. These statistics allow one to estimate the free energy of binding directly through counting. [34] The free energy of binding was obtained according to equation 1.
where the probabilities to find the system in the bound and unbound states, pb and pu, respectively, are given by the fractions of the total simulation time that the guest is found inside and outside of the cage, respectively, R is the gas constant, T is the temperature, V is the volume of the simulation box, and V Ref is a reference volume. Using a reference volume that corresponds to the chemical standard state concentration of 1 mol/l (V Ref = 1.66 nm 3 ) yields the standard-state binding free energy. The bound state was defined based on the distance between the center-of-mass of the cage to the P-atom of the diphenylphosphate guest. A histogram of the distance distribution obtained from the total 50 μs of simulation time for the cage [Pd2L 1 4+ is shown in Figure S141. The bound state was defined up to a distance of 1.4 nm, where the distribution has a minimum. The statistical uncertainties were estimated from the standard deviation of the DG's computed from the 10 individual 5 µs simulations. Figure S140: Distribution of the distance between the center-of-mass of the cage and the P-atom of the guest.
In addition to the above simulations with guest G 3 , we carried out MD simulations of the diethyl phosphate guest G 4 with the methylated cage [Pd2L 2

2L
A 2] 4+ (note that no data is given for G 4 with [Pd2L 1

2L
A 2] 4+ , since a large kinetic barrier for unbinding of the diethylphosphate guest from the cage in the MD simulations prohibited the extraction of statistically reliable results). Ten individual simulations of length 1.2 µs were carried out (all other simulation parameters as well as the simulation setup were analogous to the previous simulations described above). In total, 31 binding events were observed during the accumulated simulation time of 12 µs, again allowing one to estimate the binding free energy with statistical precision (from equation 1). For the smaller G 4 guest, the distance criterion to separate the bound and unbound states (see above) was 0.6 nm.
To more closely analyse the binding mode of the guests G 3 and G 4 to the cage, only the chunks of the MD trajectories in which the guest was bound inside the cage cavity were analysed. The likelihood to find particular interactions between the different chemical moieties of the cage and the guest, as given by the percentages in Figure 5 in the main text, are the fraction of the time where the indicated interactions were found to be present. An H-bond between cage and guest was defined if the distance between donor (cage NH group) and acceptor (guest phosphate group) was smaller than 0.35 nm and the hydrogen -donor -acceptor angle was below 30 degrees. The cage can also interact with the guest through nonpolar interactions, for example between the π-surfaces. These interactions are possible also with the methylated cage. To quantify the nonpolar contacts in our MD simulations, contacts between the cage and the two phenyl rings of the guest were counted in the bound state. A contact was defined if the distance between the center-of-mass of 3 atoms forming one ring of the cage (i.e., the ligands) and the center-of-mass of 3 atoms in the phenyl rings of the guest was closer than 0.6 nm. For all multi-ring ligands of the cage, the nonpolar contacts were counted according to the previously mentioned conditions for each ring separately and then summed over all rings that constitute the full ligand. Several simultaneous contacts of neighboring rings with the guest were counted as only one contact. Note that the contact percentages (given in Figure  5 in the main text) do not necessarily sum up to 100%, first because the different contacts are not mutually exclusive and second because the guest can be inside the cage (i.e., bound) but transiently not form any H-bond or nonpolar contact. In addition to the contacts between the structural components of the host and the guest, the interaction energies of each of these components with the guests G 3 and G 4 were calculated to take a closer look at the strength of these interactions. For that, energy groups were defined in Gromacs for each structural element of the cage, and the short-range Coulomb and Lennard-Jones interactions with the guest were calculated for each time step and then summed up and averaged over the simulation chunks were the guest was bound. The results are shown in Tables S4 and S5 for G 3 and G 4 , respectively. (with endohedral NMe) with the guest G 3 . Interactions with the solvent and the BF4 − counter ions were not considered for this calculation. LIC refers to the orange colored structural element in Figure 5 (excluding the NH/N-methyl group, whose interactions are analyzed separately), LPL refers to the red part, Py to the green part and Q to the blue part. NH / N-methyl refers to the NH group (respectively the methyl group) in the LIC element. cage, which shows that the ligand partly compensates the lack of the H-bond through stronger nonpolar contacts, which it can establish if it is not in an H-bonded configuration. The pyridine rings (Py, green) also interact strongly with the guest through non-polar contacts, showing that they also contribute to the guest binding. This interaction also becomes stronger in the absence of the H-bond. Taken together, this interaction energy analysis shows that the H-bond indeed strengthens the guest binding in this heteroleptic cage, and that the other, non-H-bonding ligand, also contributes to the binding and partly compensates the absence of the H-bond in the methylated form of the cage. (with endohedral NMe) with the guests G 3 and G 4 . Interactions with the solvent and the BF4 − counter ions were not considered for this calculation. LIC refers to the orange-colored structural element in Figure 5 (excluding the NH/N-methyl group, whose interactions are analyzed separately), LPL refers to the red part, Py to the green part and Q to the blue part. NH / N-methyl refers to the NH group (respectively the methyl group) in the LIC element.